Cholesterol is an important component of cellular membranes and is also a precursor in the synthesis of bile acids, lipoproteins and steroid hormones in certain cells. However, the synthesis of cholesterol must be carefully controlled since its excess can lead to pathologic states such as atherosclerosis. The rate limiting enzyme in cholesterol synthesis, hydroxymethylglutaryl (HMG) CoA reductase, is controlled by a bicyclic phosphorylation system involving two cAMP-independent protein kinases and three protein phosphatases. In this system, HMG-CoA reductase is phosphorylated (inactivated) by HMG-CoA reductase kinase which is itself only active after phosphorylation by HMG-CoA reductase kinase kinase. This regulatory system appears to play an inportant role in the acute control of hepatic cholesterol synthesis in response to insulin and glucagon. However, the molecular basis for this regulation remains unclear and it is the primary goal of this proposed project to elucidate these mechanisms. In previous work by the principal investigator, the protein phosphatases involved in the bicyclic phosphorylation system have been identified and extensively characterized. The experimental approach will thus involve the purification and characterization of HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase. Further experiments comparing the in vitro and in vivo sites of phosphorylation of HMG-CoA reductase and HMG-CoA reductase kinase will then be carried out to determine whether HMG-CoA reductase kinase and HMG-CoA reductase kinase kinase are the only protein kinases which phosphorylate these two enzymes, respectively, in vivo. If other protein kinases are found to be involved, these enzymes will be characterized and the effects of these new phosphorylation reactions on the activities of HMG-CoA reductase and HMG-CoA reductase kinase will be determined. These studies will provide the basis for further experiments aimed at identifying which protein kinase(s) and/or protein phosphatase(s) in the system are regulated by insulin and glucagon and at elucidating the specific biochemical mechanisms involved. The proposed studies should contribute to our understanding of the molecular basis of hormone action and of the mechanisms for regulating cholesterol synthesis. This knowledge may be useful in the design of rational approaches for pharmacological intervention in the pathogenesis of atherosclerosis.